Novel secretion modification region (SMR) peptide exhibits anti-metastatic properties in human breast cancer cells

Breast cancer is the second leading cause of cancer-related mortality in women worldwide, with nearly 90% attributed to metastatic progression. Exosomes containing epithelial–mesenchymal transition (EMT) ‘programs’ transmit pro-metastatic phenotypes. Our group discovered and developed a novel anti-cancer SMR peptide that antagonizes breast cancer cell exosome release resulting in cell cycle arrest and tumor growth suppression. This study aims to evaluate the anti-metastatic capabilities of the SMR peptide, focusing on exosomes and EMT. Breast cancer cell lines MDA-MB-231 and MCF-7 were treated with the SMRwt peptide, and the following assays were performed: cell wound-healing, migration, invasion. The SMRwt peptide consists of the following amino acid sequence VGFPVAAVGFPVDYKDDDDK and contains the SMR domain (66VGFPV70) of the HIV-1 Nef protein. Western blot analysis detected epithelial and mesenchymal markers to evaluate EMT progression. Extracellular vesicle type and quantity were assessed through NanoSight analysis. Mortalin and Vimentin knockdown was achieved through antibody targeting and miRNAs. Data gathered demonstrated that the SMR peptide interacts with Mortalin and Vimentin to inhibit pro-EMT exosome release and induce EMT tumor suppressor protein expression. Specifically, SMRwt treatment reduced mesenchymal markers Mortalin and Vimentin expression, while the epithelial marker E-cadherin expression was increased in breast cancer cells and breast cancer-derived exosomes. The SMR peptide specificity was identified as no effect was observed for MCF-10A exosome release or function. Direct Mortalin knockdown paralleled the results of SMR peptide treatment with an effective blockade of breast cancer cell migration. Conversely, the invasion assay differed between breast cancer cell lines with invasion blocked for in MCF-7 but not in MDA-MB-231. These results reinforce the therapeutic value of targeting breast cancer exosome release and reinforce Mortalin and Vimentin as critical regulators and therapeutic targets in breast cancer cell progression, EMT, and metastatic potential. A greater understanding of the SMR peptide mechanism of action will benefit the therapeutic design of anti-metastatic agents.


AcquisiƟon of migratory abiliƟes.
EMT program examples: TGF-β, HIF-1-α, tenascin, miR-9, miR-10b, miR-155, miR-221, miR-222 Acquisition of migratory abilities. The cargo of tumor derived EVs is frequently referred to as a pro-EMT program whose transmission throughout the tumor population drives an aggressive phenotype. (2) Directional movement. EV release of chemokines encourages movement of breast cancer cells along the path most conducive with breast cancer spread. (3) Path Creation. Degradative enzymes within EVs rapidly remove ECM to clear a path for breast cancer migration and invasion. EVs have been paralleled to 'stepping-stones' as they adhere via heparan sulfate and express fibronectin that can be bound by integrins on the surface of migrating breast cancer cells. Created with BioRender.
Therapeutic potential of SMR peptide mediated EV inhibition. Exosome-based communication networks represent a promising therapeutic target for inhibition. Our lab developed an anticancer peptide derived from the Secretion Modification Region (SMR; 66 VGFPV 70 ) of the HIV-1 Nef protein. The Nef SMR sequence interacts with cellular proteins, including mortalin and vimentin, and blocks breast cancer cell exosome release. Mortalin belongs to the heat shock protein (HSP) 70 family and acts as a highly conserved molecular chaperone encoded by the nuclear gene HSPA9B. Many studies have reported Mortalin as strongly related to the induction and/or upregulation of EMT, carcinogenesis, and metastasis [31][32][33][34][35][36] . Mortalin is known to trigger pro-metastatic tumor derived exosome release. However, the specific mechanism of Mortalin's role in regulating endosomal trafficking remains unclear [37][38][39][40] . Vimentin is a 57 KD mesenchymal marker type III intermediate filament that maintains cell integrity and is involved in cell migration, motility, and adhesion. Overexpression of Vimentin in solid cancers drives EMT, invasion, and metastasis 4 . In vitro studies have demonstrated that the knockdown of Vimentin impairs cell attachment, migration, and invasion in breast and colon cancer cell lines 41 . In our previous research, SMR peptide targeting antagonized breast cancer cell proliferation, arrested the cell cycle, and restored complement mediated cell death 42 . To better understand the therapeutic potential of the SMR peptide, we aim to explore the mechanism of SMR mediated inhibition on breast cancer EMT, invasion, and migration.

Materials and methods
Cell cultures and antibodies. Cell  Peptides. Our previous research showed that an extensive genetic analysis of Nef revealed several highly conserved N-terminal domains that were necessary and sufficient for Nef-induced exNef secretion. One of these domains, the novel SMR ( 66 VGFPV 70 ), is highly conserved across all HIV-1 clades, HIV-2, and SIV. A peptide containing the SMR sequence (SMRwt; described in 43 ) was synthesized. A control peptide, SMRmut, in which valine-65 was replaced by alanine (AGFPV) was a negative control (SMRmut; described in 43 ). Both SMRwt and SMRmut peptides were custom-made by InnoPep Inc. (San Diego, CA, USA). In addition, modified forms of the SMRwt and SMRmut peptides were generated as follows: SMRwt-CPP, PEG-SMRwt-CLU, SMRmut-CPP, and PEG-SMRmut-CLU (described in 42 ). CPP is a cell-penetrating peptide derived from HIV-1 tat protein (C-terminus), PEG is poly-ethylene-glycol (N-terminus), and CLU is clusterin (C-terminus).
Breast cancer peptide uptake. MDA-MB-231 and MCF-7 breast cancer cells were treated with SMR peptides using the Chariot™ reagent (Active Motif, Carlsbad, CA, USA), as previously described 43  Co-culture and tumor cell transendothelial migration assay. The transendothelial migration of breast cancer cells was detected using CytoSelectTM (Cell Biolabs, Inc., San Diego, CA, USA) following the manufacturer's instructions. The transwell cell culture chamber with polycarbonate filters (8 µm pore size; 0.33 cm 2 area) This co-culture system mimics the in vivo orientation of endothelial and tumor cells during tumor cell intravasation tumor cells to make close contacts with HUVEC basal pole. 1 × 10 5 HUVEC were seeded first on the lower side of the Transwell filter and grown to confluence for 48 h. 2 × 10 5 of the overnight serum-starved transfected breast cancer cells were labeled with a fluorescent dye seeded to the monolayer of the HUVEC cells.
The insert was then transferred to a new plate containing a fresh medium with 10% fetal bovine serum. The assay was performed at 37 °C for 24 h. non-migrating cells at the top were removed, whereas cells that migrated to the bottom of the membrane were first dissociated from the membrane, then lysed and quantified using CyQuant GR fluorescent dye 480 nm/520 nm.
Transwell migration and invasion assays. Transwell migration and invasion assays were performed as Creative Bioarray described (https:// www. creat ive-bioar ry. com/ trans well-migra tion-and-ivasi on-assays. htm). Note: for the invasion assay, Matrigel (1 mg/mL) was added to the upper compartment of the transwell membrane inserts with 8 µm pore size polycarbonate filters (Millipore-Sigma, CA, USA) and solidified through incubation at 37 °C for 2 h. 1 × 10 6 breast cancer cells were treated overnight with different SMR peptide dosages (in µM) as follows: 0, 0.035, 0.7, 0.14, 0.28, 0.56, 1.12, 2.24. Next, breast cancer cells (1 × 10 6 cells per well) were resuspended in 100 µL of FBS-free media and seeded to the top of the transwell membrane inserts. RPMI 1640 media with 10% FBS was added to the lower compartment, and the plate was incubated at 37 °C for 24 h. The cells that migrated/invaded the lower surface of the membrane were fixed with 70% ethanol and stained with crystal violet for viewing using a 10 × objective under an inverted microscope.
Exosome isolation and purification. Exosomes were isolated from breast cancer cells by established differential centrifugation and ultracentrifugation 42 . Untreated or treated SMRmut peptide breast cancer cells were used as controls. Treated and untreated breast cancer cell culture supernatants underwent sequential centrifugations as follows: 400×g for 10 min and 10,000×g for 30 min, and 200,000×g for 2 h. The pelleted exosomes were washed, resuspended with PBS, and stored at 4 °C until use for Nanosight analysis.
Nanoparticle tracking analysis measurement with Nanosight NS300. All (Fig. 3A,B). The IC50 for MCF-7 migration inhibition was 0.082 µM (95% CI 0.033-0.240) (Fig. 3A,B). In contrast, the invasion assay differed between cell lines with MCF-7 invasion effectively inhibited and MDA-MB-231 invasion unaffected by the presence of the SMR peptide within the dose levels administered (Fig. 3C,D). A simple linear regression revealed that the MDA-MB-231 invasion data did not significantly differ from a zero slope, reaffirming the lack of impact SMRwt peptide had on the invasion capability (Fig. 3C). In addition, an ANOVA with multiple comparisons was performed to compare the mean MDA-MB-231 invaded cell count for the control to that of the different SMR peptide dose conditions, but only one significant difference was identified at an SMR peptide concentration of 0.28 μM (p-value < 0.05) (Fig. 3C). MCF-7 invasion was strongly inhibited with increasing dosages of SMRwt peptide. Like the migration assay for MCF-7, the invasion assay had near elimination of invasion at the highest dosage level (2.24 µM) (Fig. 3D). Nonlinear regression analysis for MCF-7 analysis determined the following equation: Y = − 37.53 + [195.5 − (− 37.53)]/ [1 + (X/IC50)] with an R 2 value of 0.912 (Fig. 3D). The IC50 for MCF-7 invasion inhibition was 0.315 µM (95% CI 0.098-1.457) (Fig. 3D). In addition, an ANOVA with multiple comparisons was performed to compare the mean MCF-7 invaded cell count for the control to that of the different SMR peptide dose conditions. Significant differences were identified at the following SMR peptide concentrations (in μM) 0.28 (p-value < 0.005), 0.56 (p-value < 0.0005), 1.12 (p-value < 0.00005), and 2.24 (p-value < 0.00005) (Fig. 3D). As the invasion assay for MCF-7 required a higher dosage to attain inhibition with the SMRwt peptide than the migration assay, the MDA-MB-231 cells may have invasion be inhibited at high SMRwt peptide dosages.

MCF-7 E-Cadherin
RelaƟve Band Intensity (%)   These results reinforce the therapeutic value of targeting breast cancer exosome release and reinforce Mortalin and Vimentin as critical regulators and therapeutic targets in breast cancer cell progression, EMT, and metastatic potential. A greater understanding of the SMR peptide mechanism of action will benefit the therapeutic design of anti-metastatic agents.

Discussion
Tumor cell acquisition of a migratory mesenchymal phenotype and invasion into the surrounding matrix are early events of metastasis occurrence. Proteins, such as Mortalin and Vimentin, which contribute to this early pathological migration and invasion process may be critical molecular targets for early breast cancer metastasis intervention. Recent reports indicated a correlation between Mortalin expression level with metastatic potential and tumor recurrence, suggesting the clinical application of Mortalin as a chemotherapeutic drug target. Mortalin expression levels correlate with the development of epithelial-mesenchymal-transformation (EMT), a crucial step for metastasis and inactivation of tumor suppressor p53 protein, deregulation of apoptosis, and activation of EMT signaling 31 . High levels of Mortalin in breast cancer are associated with the mesenchymal markers, whereas the epithelial markers are downregulated 44 . Malignant cells undergoing EMT acquired migration and drug resistance characteristics 31,45 . EMT is a process characterized by: (a) the absence of polarity and intercellular adhesion of epithelial cells, (b) the acquisition of mesenchyme features associated with higher motility, and (c) the altered expression of EMT biomarkers (down-regulation of epithelial marker E-cadherin and up-regulation of the mesenchymal marker Vimentin). Mortalin is upregulated in human breast cancer cells. Its depletion robustly induces cell death and growth arrest in breast cancer cell lines in culture and can interact with p53, affecting the cell cycle and survival 46,47 . Anti-tumor peptides, such as the SMRwt peptide, also induce tumor cell arrest at cell cycle G2/M in MDA-MB-231 and MCF-7 breast cancer cells 42 . In addition, the regulation of invasive and migratory properties for carcinomas involves many factors that contribute to complex intercellular crosstalk and networking 35,48 . An autocrine loop exists for the vascular endothelial growth factor (VEGF) to induce cell migration and invasion of breast cancer cells. MCF-7 cells express lower levels of VEGF than MDA-MB-231 cells, which have high invasive and migration capacities 49 . Without estrogen supplementation, MCF-7 cells do not induce metastasis in mice and have a low migration capacity in vitro 50 . Youngs et al. 51 demonstrated a dosedependent migratory response of breast cancer cells to increasing concentrations of exogenous CCL2. The Tumor cell acquisition of a migratory mesenchymal phenotype and invasion into the surrounding CCL2 expression has been shown to correlate with progression in pancreatic cancer 52 and breast cancer 53 . This study evaluated whether SMRwt peptide inhibition of breast cancer cell migration and invasion could effectively inhibit the growth of human breast cancer cells and whether the anti-tumor effect was linked to the reduction of tumor exosomes secretion. The results indicate that breast cancer cells treated with the SMRwt peptide displayed decreased function of the human chaperone protein Mortalin, significantly affecting tumor cell proliferation and reducing tumor cell invasion and migration. These results further support the role of Mortalin in breast cancer progression and support the role of the SMRwt peptide as an antagonist to Mortalin function. The SMRwt peptide also antagonizes EV release. The SMRwt peptide reduces the expression of Mortalin, which could play a role in breast cancer cell invasion and metastasis, thus suggesting potential peptide applications in early-stage breast cancer chemotherapy. This study has also demonstrated that the SMRwt peptide plays a novel and crucial role in breast cancer metastasis migration and invasion. Knockdown of Mortalin and Vimentin leads to E-cadherin expression changes, which facilitate adhesion formation and reduce Vimentin, a critical transcription factor in EMT 54 . Furthermore, present data suggest that SMRwt peptide could block breast cancer metastasis through mechanisms involving target protein Mortalin and Vimentin that decrease proliferation, tumorigenesis, and tumor exosome release. Therefore, SMRwt peptide could be a potential novel strategy for breast cancer therapy, especially in early-stage breast cancer patients.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.